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Site-directed mutagenesis studies of conserved metal-binding residues in DmpFG, a bifunctional aldolase/dehydrogenase from Pseudomonas sp. strain CF600

Title:

Site-directed mutagenesis studies of conserved metal-binding residues in DmpFG, a bifunctional aldolase/dehydrogenase from Pseudomonas sp. strain CF600

Tait, Andrew (2004) Site-directed mutagenesis studies of conserved metal-binding residues in DmpFG, a bifunctional aldolase/dehydrogenase from Pseudomonas sp. strain CF600. Masters thesis, Concordia University.

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Abstract

DmpFG is bifunctional aldolase/dehydrogenase which catalyzes the last two steps of the meta -cleavage pathway of catechol in Pseudomonas sp. strain CF600. 4-Hydroxy-2-ketovalerate undergoes divalent cation-dependent aldol cleavage to yield pyruvate and acetaldehyde, and the acetaldehyde is then oxidized to acetyl-CoA in an NAD + - and CoA-dependent reaction. Because of its unique primary sequence, structure, and requirement for Mn 2+ , DmpG (aldolase) apparently represents a new sub-class of class II aldolases. X-ray crystallography of DmpFG identified His200 and His202 of a conserved HXH motif in DmpG as potential active-site residues; these histidines were observed to be complexed with a metal ion, which in turn was complexed with a molecule of substrate-analogue (Manjasetty, B. A., Powlowski, J., Vrielink, A. (2003) Proc. Natl. Acad. Sci. 100(12) 6992-6997). To evaluate the structural and functional importance of His200 and His202 of DmpG, H200A, H202A, H203A and H202AH203A variants were constructed, expressed and purified. (Abstract shortened by UMI.)

Divisions:Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry
Item Type:Thesis (Masters)
Authors:Tait, Andrew
Pagination:xiii, 95 leaves : ill. ; 29 cm.
Institution:Concordia University
Degree Name:M. Sc.
Program:Chemistry and Biochemistry
Date:2004
Thesis Supervisor(s):Powlowski, Justin
ID Code:8149
Deposited By:Concordia University Libraries
Deposited On:18 Aug 2011 14:16
Last Modified:18 Aug 2011 15:44
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